Pathological mutations reveal the key role of the cytosolic iRhom2 N-terminus for phosphorylation-independent 14-3-3 interaction and ADAM17 binding, stability, and activity.

The protease ADAM17 plays an important role in inflammation and cancer and is regulated by iRhom2. Mutations in the cytosolic N-terminus of human iRhom2 cause tylosis with oesophageal cancer (TOC). In mice, partial deletion of the N-terminus results in a curly hair phenotype (cub). These pathological consequences are consistent with our findings that iRhom2 is highly expressed in keratinocytes and in oesophageal cancer. Cub and TOC are associated with hyperactivation of ADAM17-dependent EGFR signalling. However, the underlying molecular mechanisms are not understood. We have identified a non-canonical, phosphorylation-independent 14-3-3 interaction site that encompasses all known TOC mutations. Disruption of this site dysregulates ADAM17 activity. The larger cub deletion also includes the TOC site and thus also dysregulated ADAM17 activity. The cub deletion, but not the TOC mutation, also causes severe reductions in stimulated shedding, binding, and stability of ADAM17, demonstrating the presence of additional regulatory sites in the N-terminus of iRhom2. Overall, this study contrasts the TOC and cub mutations, illustrates their different molecular consequences, and reveals important key functions of the iRhom2 N-terminus in regulating ADAM17.

Friedel-crafts alkylation of acylphloroglucinols catalyzed by a fungal indole prenyltransferase.

Naturally occurring prenylated acylphloroglucinol derivatives are plant metabolites with diverse biological and pharmacological activities. Prenylation of acylphloroglucinols plays an important role in the formation of these intriguing natural products and is catalyzed in plants by membrane-bound enzymes. In this study, we demonstrate the prenylation of such compounds by a soluble fungal prenyltransferase AnaPT involved in the biosynthesis of prenylated indole alkaloids. The observed activities of AnaPT toward these substrates are much higher than that of a microsomal fraction containing an overproduced prenyltransferase from the plant hop.

AstPT catalyses both reverse N1- and regular C2 prenylation of a methylated bisindolyl benzoquinone.

Prenylated bisindolyl benzoquinones exhibit interesting biological activities, such as antidiabetic or anti-HIV activities. A number of these compounds, including asterriquinones, have been isolated from Aspergillus terreus. In this study, we identified two putative genes by genome mining, ATEG_09980 and ATEG_00702, which share high sequence similarity with the known bisindolyl benzoquinone prenyltransferase TdiB from Aspergillus nidulans. The coding sequences were cloned and overexpressed in E. coli. The overproduced recombinant proteins were purified to near homogeneity and used for enzyme assays with asterriquinone D in the presence of dimethylallyl diphosphate. HPLC analysis showed that product formation was only detected in enzyme assays with EAU29429 encoded by ATEG_09980, not in those with EAU39348 encoded by ATEG_00702. Product isolation and structure elucidation by NMR and MS analyses led to identification of N1-reversely and C2-regularly monoprenylated derivatives, as well as N1',N1''reversely, N1'-reversely, C2''-regularly diprenylated derivatives. This proved that EAU29429 functions as an asterriquinone prenyltransferase (AstPT) and indicated the involvement of EAU29429 rather than EAU39348 in the biosynthesis of methylated asterriquinones. Furthermore, incubation of monoprenylated enzyme products with AstPT resulted in the formation of the diprenylated derivatives.

New insights into the biosynthesis of prenylated xanthones: Xptb from Aspergillus nidulans catalyses an O-prenylation of xanthones.

Gene-inactivation experiments have indicated that the putative prenyltransferase XptB from Aspergillus nidulans was likely to be responsible for the prenylation of 1,7-dihydroxy-6-methyl-8-hydroxymethylxanthone. Recently, it was suggested that this enzyme might also accept as substrate the benzophenone arugosin H, which is assumed to be a precursor of prenylated xanthones. In this study, five benzophenones and ten xanthones were incubated with purified recombinant XptB in the presence of dimethylallyl diphosphate (DMAPP). XptB accepted four xanthones as substrates, including the proposed natural substrate, and catalysed regiospecific O-prenylations at C-7 of the xanthone core. K(m) values in the range of 0.081-1.1 mM and turnover numbers (k(cat)) between 0.02 and 0.5 s(-1) were determined for the accepted xanthones. The kinetic parameters for DMAPP were found to be 0.024 mM (K(m)) and 0.13 s(-1) (k(cat)). Arugosin H was not accepted by XptB under the tested conditions. XptB was relatively specific towards its prenyl donor and did not accept geranyl or farnesyl diphosphate as substrate. Mn(2+) and Co(2+) strongly enhanced XptB activity (up to eightfold); this has not been reported before for prenyltransferases of the DMATS superfamily.

Breaking the regioselectivity of indole prenyltransferases: identification of regular C3-prenylated hexahydropyrrolo[2,3-b]indoles as side products of the regular C2-prenyltransferase FtmPT1.

The prenyltransferase FtmPT1 from Aspergillus fumigatus is involved in the biosynthesis of fumitremorgin-type alkaloids and catalysed the regular C2-prenylation of brevianamide F (cyclo-L-Trp-L-Pro). It has been shown that FtmPT1 also accepted a number of other tryptophan-containing cyclic dipeptides and prenylated them, in the presence of dimethylallyl diphosphate, at C-2 of the indole nucleus. Detailed analysis of the incubation mixtures of FtmPT1 with these cyclic dipeptides revealed the presence of additional product peaks in the HPLC chromatograms. Seven regularly C3-prenylated hexahydropyrrolo[2,3-b]indoles were isolated and identified by HR-ESI-MS and NMR analyses including HMBC, HMQC and NOESY experiments. Further experiments proved that the C2- and C3-prenylated products are both independent enzyme products. To the best of our knowledge, this is the first report on the enzymatic formation of regularly C3-prenylated indolines. A reaction mechanism for both C2- and C3-prenylated derivatives was proposed.

Prenylation at the indole ring leads to a significant increase of cytotoxicity of tryptophan-containing cyclic dipeptides.

Fourteen tryptophan-containing cyclic dipeptides 1a-14a, including all four stereoisomers of cyclo-Trp-Pro and cyclo-Trp-Ala, were converted to their C2-regularly prenylated derivatives 1b-14b in the presence of dimethylallyl diphosphate by using the purified recombinant FtmPT1 as catalyst. The enzyme products were isolated on HPLC in preparative scales and their structures were elucidated by NMR and MS analyses. The cytotoxic effects of the prenylated products and their substrates were tested with human leukemia K562 and ovarian cancer A2780 sens and A2780 CisR cell lines. Preliminary results have been clearly shown that prenylation at C2 led to a significant increase of the cytotoxicity of the tested cyclic dipeptides in all the 14 cases. The second amino acid and the stereochemistry of tryptophan moiety of the cyclic dipeptides showed less influence on the cytotoxicity of the tested compounds.